ThermoGen proposes to develop an in vivo protein engineering system that will allow rapid, random shuffling of protein domains to produce novel protein fusions. The proposed system, based on the phase I advancements, will be generally applicable to production of multifunctional chimeric proteins and will allow functional domain mapping to probe protein structure. The system will complement in vitro tools that are commonly used in protein design. Phase I involved constructing and successfully testing transposon elements that show the feasibility of creating both carboxy- and amino- terminal protein fusions in vivo. Phase II will incorporate new features into the system that make it both easier to use and more versatile. Refinement and optimization of the in vivo system also will be performed. Several protein domains commonly used in gene fusion applications will be coupled with the system and used to create fused protein products with proprietary ThermoGen enzymes. In addition, construction of multifunctional fusion proteins with commercial applications will be explored. Phase III will involve licensing or contracting work with the elements, packaging it as a kit for general use, and marketing the fusion products made with the in vivo protein engineering system.